Comparison And Experimental Study Of Immune Tolerance Mice Models Of Allergic Conjunctivitis

Objective:The mice model of immune tolerance of allergic conjunctivitis was constructed and comparatived by different methods.Method:50 Balb/c newborn mice were randomly divided into blank control group(Blank),OVA+subcutaneous injection group,OVA+aerosol inhalation group,OVA+gastric group,RW+subcutaneous injection group,RW+aerosol inhalation group,RW+gastric group,HDM+nebulized inhalation group,HDM+intragastric group(n=5 animals/group).With the exception of the blank control group,mice in each group were individually exposed to the corresponding antigens to induce immune tolerance early in life and stimulated with the corresponding antigens in adulthood.After the final ocular challenge,the ocular surface was visualized using anterior segment photography,all the mice were sacrificed 24h later,and the expression of conjunctival RANTES and IL-17 mRNA was measured by RT qPCR and serum IL-17 concentration was measured by ELISA.Results:The OVA+nebulized group,OVA+gavage group,HDM+subcutaneous group showed difficulty in opening eyes under strong light;mice in the RW+nebulized group could see filamentous mucus secretions at the cornea and bulbar conjunctiva.In RW+gavage group,the mice showed a large amount of mucus secretions that caused lower eyelid eyelashes to fall into the cornea and bulbar conjunctiva;OVA+subcutaneous group,RW+subcutaneous group,HDM+ nebulized group had relatively light ocular surface signs.The expression level of IL-17 mRNA in conjunctival tissue was the highest in the RW+gavage group,and the expression level of inhibition was more significant in the OVA+subcutaneous group and RW+subcutaneous group.The conjunctival tissue RANTES mRNA level was highest in the RW+gavage group,and the expression level was suppressed more significantly in the RW+subcutaneous group and HDM+ subcutaneous group.Results from the Elisa showed that the serum IL-17 concentration of the mice in the OVA+gavage group,the RW+gavage group and the HDM+nebulization group was the highest,while the IL-17 concentration in the serum of the OVA+nebulization group and the RW+subcutaneous group was more significantly inhibited.Conclusion:The immune tolerance of conjunctival tissue and systemic immune tolerance were consistent.Subcutaneous injection is a more appropriate method to construct an immune tolerance model.Objective:A mice model of allergic conjunctivitis was constructed by ovalbumin,ragweed pollen and house dust mites,respectively,and their sensitization effects were compared and to select the most sensitive antigen.Method:20 SPF BALB/c newborn mice were randomly divided into four groups:negative control group,ovalbumin(OVA)group,ragweed pollen(RW)group and house dust mite(HDM)group(n=5 mice/group).Anterior segment photography was used to directly observe changes in the ocular surface of mice in each group;RT-qPCR was used to detect Chemokine ligand 5(RANTES)and IL-17 in the conjunctival tissue of mice;Elisa was used to detect IL-4,IL-5,IL-13 and IL-17 concentration changes in serum.Results:In the NC group,the eyelids were not swollen,the blepharoplasty was normal,the conjunctiva was not congested or edema,and no obvious secretion was produced in the conjunctival sac.In OVA group,RW group and HDM group showed obvious eyelid swelling,conjunctival hyperemia and edema,and mucous secretions soaked the conjunctival sac and the hair around the eyes.Among them,the OVA group and the RW group mice had difficulty opening eyes,and closed eyes with strong light reflection.RT-qPCR results showed that the relative expression levels of conjunctival tissue IL-17 and RANTES mRNA were the highest in RW group.However,the results of Elisa showed that the concentration levels of Th2 cytokines(IL-4,IL-5,IL-13)in the OVA,RW,and HDM groups were similar,while the concentration of IL-17 was the highest in the RW group.Conclusion:Different antigens can induce the occurrence of allergic conjunctivitis in mice,and the expression levels of RANTES mRNA and IL-17 mRNA in the conjunctival tissues of mice induced by ragweed pollen have the most significant increase in serum IL-17 concentration.Therefore,we believe that ragweed pollen should be more allergenic.Objective:We explored the expression of Th17/Treg cells in the mice model of allergic conjunctivitis immune tolerance.Method:15 SPF grade BALB/C newborn mice were randomly divided into three groups:Control(NC)group,Positive control group(PC)group and Immune tolerance(IT)group(n=5 mice/group).The changes of ocular surface were observed by anterior segment photography 20 minutes after the model was established successfully,and the mice were sacrificed after 24 hours.The number of mast cells was observed by HE staining.The expression of RANTES was observed by immunohistochemistry.The expression of IL-17 mRNA and Foxp3 mRNA were observed by RT-qPCR.The changes of spleen index were observed.The spleen single cell suspension was prepared to detect the changes of IL-17 and CD4+CD25+by flow cytometry.The supernatant of spleen single cell suspension and serum were tested by Elisa for Total-IgE and IL-10.IL-17 expression.Results:There was no allergic conjunctivitis in NC group.In PC group,eyelid swelling,difficulty in opening eyes,bulbar conjunctival edema and large amount of mucinous secretion in conjunctival sac were observed in PC group.In the IT group,the eyelids were slightly swollen,a small amount of secretion in the conjunctival sac,and some eyelashes were in contact with conjunctiva and cornea.The results of immunohistochemistry told us(100X)that a large amount of RANTES was expressed in the intercellular substance and epithelial layer in the PC group,a small amount of RANTES positive staining was seen in the IT group,and no RANTES positive staining was seen in the NC group.Although the expression level of IL-17 mRNA in the IT group was suppressed,it was still between the NC and PC groups.But interestingly,the expression level of Foxp3 mRNA in the IT group was significantly higher than that in the NC and PC groups.The results from Elisa showed that the concentration of IgE in the serum and spleen supernatant of the IT group was between the NC group and the PC group;while the IL-10 concentration in the serum and spleen supernatant of the IT group was close to that of the NC group,but significant Higher than the PC group;the concentration of IL-17 in the serum and splenocyte supernatant of the IT group is also between the NC group and the PC group,and its expression trend is similar to IgE.The results of flow cytometry showed that the percentage of IL-17 in the IT group was higher than that in the NC group but lower than that in the PC group;the percentage of CD4+CD25+in the IT group was significantly higher,while it was inhibited in the PC group.Conclusion:Mice exposed to multiple antigens in the early stage of life can inhibit the conjunctival reaction caused by repeated exposure to the same allergen in adulthood and enhance the immune tolerance.The formation of immune tolerance in mice is related to the state of Th17 and Treg cells.The activity of Treg cells was increased and Th17 cells were inhibited in the immune tolerance of allergic conjunctivitis.Objective:To study the expression of proinflammatory factors(RANTES,Total-Ige,IL-17)and anti-inflammatory factors(IL-10,CD4+CD25+)in immune tolerant mice with allergic conjunctivitis under the regulation of NF-kB/P-65 signaling pathway.Method:30 SPF grade Balb/c newborn mice,half male and one female,were randomly divided into NC+PBS group,NC+MA group,PC+PBS group,PC+MA group,IT+PBS group,IT+MA group(n=5/group).20 minutes after the last eye drop,the anterior segment was photographed,and the mice were killed 24 hours later.The P-65(cytoplasmic and nuclear),IKB? and pIKB? were detected by Western blot The expression of RANTES in conjunctiva was detected by immunohistochemistry;the concentrations of total IgE,IL-10 and IL-17 in serum and supernatant of spleen cells were detected by ELISA;the expression of IL-17 and CD4+CD25+in spleen single cell suspension was detected by flow cytometry.Results:The activation level of NF-kB in the NC+PBS and NC+MA groups was low,while the activation level in the PC+PBS and IT+PBS groups was higher than that in the PC+MA and IT+MA groups.There was no significant difference in ocular surface condition between NC+PBS group and NC+MA group.Compared with the PC+PBS group,the PC+MA group did not improve the eye surface conditions except for the improvement of eye opening in bright light.Compared with the IT+PBS group,the ocular surface of the IT+MA group was significantly improved,there was no obvious swelling of the eyelids in the mice,no edema in the bulbar conjunctiva,but a small amount of mucus secretions were still seen in the conjunctival sac.We also can find from the results of immuno-histochemistry(100X)that,except for the NC+MA group,the positive staining of the mice in the other MA+group was decreased.Our Elisa results showed that after MA treatment,the concentration of IL-10 in the spleen supernatant and serum is close to the concentration of PBS treatment,and it was not significantly affected by MA.But the difference was that the IL-17 concentration of mice after MA treatment was decreased.Although the IgE concentration also was decreased,the consistency of expression in the spleen supernatant and serum was lower.The results of flow cytometry showed that the percentage of IL-17 in the PC+MA group and IT+MA group was lower than that in the PC+PBS group and IT+MA group.The percentage of CD4+CD25+in the MA treatment group and the PBS group were closely.Conclusion:After the NF-kB signaling pathway was inhibited,the immune tolerance of mice was enhanced.Once NF-kB was inhibited,the activity of Treg cells was not significantly affected,but the function of Th17 cells was decreased and the immune tolerance of mice was enhanced,which indicated that the function of Th17 cells was more closely related to the immune tolerance of mice.